Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
  • A01H6/4678—Triticum sp. [wheat]
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
  • A01H5/10—Seeds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B07—SEPARATING SOLIDS FROM SOLIDS; SORTING
  • B07C—POSTAL SORTING; SORTING INDIVIDUAL ARTICLES, OR BULK MATERIAL FIT TO BE SORTED PIECE-MEAL, e.g. BY PICKING
  • B07C5/00—Sorting according to a characteristic or feature of the articles or material being sorted, e.g. by control effected by devices which detect or measure such characteristic or feature; Sorting by manually actuated devices, e.g. switches
  • B07C5/34—Sorting according to other particular properties
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
  • C12N15/8289—Male sterility
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B07—SEPARATING SOLIDS FROM SOLIDS; SORTING
  • B07C—POSTAL SORTING; SORTING INDIVIDUAL ARTICLES, OR BULK MATERIAL FIT TO BE SORTED PIECE-MEAL, e.g. BY PICKING
  • B07C5/00—Sorting according to a characteristic or feature of the articles or material being sorted, e.g. by control effected by devices which detect or measure such characteristic or feature; Sorting by manually actuated devices, e.g. switches
  • B07C5/04—Sorting according to size
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B07—SEPARATING SOLIDS FROM SOLIDS; SORTING
  • B07C—POSTAL SORTING; SORTING INDIVIDUAL ARTICLES, OR BULK MATERIAL FIT TO BE SORTED PIECE-MEAL, e.g. BY PICKING
  • B07C5/00—Sorting according to a characteristic or feature of the articles or material being sorted, e.g. by control effected by devices which detect or measure such characteristic or feature; Sorting by manually actuated devices, e.g. switches
  • B07C5/16—Sorting according to weight

Definitions

• the present invention relates to the field of agriculture, more particular hybrid seed production and separation of hybrid seed from a seed mixture using separation methods based on differences in the phenotype of the seeds comprised in said seed mixture. Separation of hybrid seeds takes place based on e. g. differences in size, surface condition or specific weight of the hybrid seeds as compared to other seeds comprised in the mixture, e. g. inbred seeds.
• Hybrid plants are bred to improve the characteristics of the resulting plants, such as better yield or yield stability, greater uniformity, improved color, disease resistance, etc..
• the commercial hybrid seed market was launched in the 1920s, with the first hybrid maize. All of the hybrid seeds planted by the farmer will be the same hybrid while the seeds from the hybrids planted will not consistently have the desired characteristics.
• Heterosis occurs in common (hexaploid) wheat, but it is difficult to produce seed of hybrid cultivars on a commercial scale (as is done with maize) because wheat flowers are bi-sexual and normally self-pollinate.
• Commercial hybrid wheat seed has been produced using chemical hybridizing agents; these chemicals selectively interfere with pollen development, or naturally occurring cytoplasmic male sterility systems.
• Hybrid plants are created by crossing a pollen parent to a seed parent of a different line or cultivar. In most cases, the male fertile pollen parent is also female fertile resulting in the production of inbred seed in addition to the hybrid seed produced on the seed parent.
• Figure 1 Kernel phenotypes in cereals.
• the free- threshing phenotype is maternally determined.
• Figure 2 Inheritance of threshability in cereals (wheat and oats) when seed parent is homozygous for one or more dominant free threshing (FT) allele(s) and the pollen parent is homozygous for one or more recessive hulled (h) allele(s), and the free-threshing phenotype is maternally expressed.
• the harvested seed from an Fl seed production field is a combination of free threshing hybrid seed from the seed parent and hulled inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow.
• the F2 grain resulting from planting of the hybrid seed is all free threshing.
• Figure 3 Inheritance of threshability in cereals (wheat and oats) when seed parent is homozygous for one ore more recessive hulled (h) allele(s) and the pollen parent is homozygous for one or more dominant free threshing (FT) allele(s), and the free-threshing phenotype is maternally expressed.
• the harvested seed from an Fl seed production field is a combination of hulled hybrid seed from the seed parent and free threshing inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow.
• the F2 grain resulting from planting of the hybrid seed is all free threshing.
• Figure 4 Inheritance of threshability in cereals (wheat and oats) when the seed parent is homozygous for one or more dominant free threshing (FT) allele(s) and the pollen parent is homozygous for one or more recessive hulled (h) allele(s), and the free-threshing phenotype is expressed in the embryo or endosperm (principle).
• the harvested seed from an Fl seed production field is a combination of free threshing hybrid seed from the seed parent and hulled inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow.
• the F2 grain resulting from planting of the hybrid seed is a combination of free threshing and hulled seeds.
• Figure 5 Inheritance of threshability in cereals (barley) when the seed parent is homozygous for one or more dominant covered (H) allele(s) and the pollen parent is homozygous for one or more recessive naked (ft) allele(s), and the free-threshing phenotype is expressed in the embryo or endosperm.
• Harvested seed from an Fl seed production field is a combination of covered hybrid seed from the seed parent and naked inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow.
• the F2 grain resulting from planting of the hybrid seed is a combination of covered and naked seed.
• Figure 6 Inheritance of threshability in cereals (barley) when the seed parent is homozygous for one or more dominant covered (H) allele(s) and the pollen parent is homozygous for one or more recessive naked (ft) allele(s), and the free-threshing phenotype is maternally expressed (principle).
• the harvested seed from an Fl seed production field is a combination of covered hybrid seed from the seed parent and naked inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow.
• the F2 grain resulting from planting of the hybrid seed is all covered.
• Figure 7 Inheritance of threshability in cereals (barley) when the seed parent is homozygous for one or more recessive naked (ft) allele(s) and the pollen parent is homozygous for one or more dominant covered (H) allele(s), and the free-threshing phenotype is maternally expressed (principle).
• the harvested seed from an Fl seed production field is a combination of covered hybrid seed from the seed parent and naked inbred seed from the pollen parent, which can be separated by sorting, as indicated by the two-way arrow.
• the F2 grain resulting from planting of the hybrid seed is all covered.
• the present invention relates to a method of separating hybrid seeds of a cereal from a mixture comprising said hybrid seeds and inbred seeds of said cereal, comprising (a) obtaining or providing a mixture of seeds of a cereal obtained or produced by interplanting a combination of plants comprising a male sterile or conditionally male sterile seed parent and a male fertile pollen parent, wherein said seed parent and pollen parent produce at least two types of seeds, at least one of which being said hybrid seeds, said at least two types of seed having different phenotypes allowing separation of said hybrid seed with non-invasive methods based on the differences in said phenotypes, said different phenotypes being determined by an allele or a combination of alleles at one or more genetic loci, wherein said seed parent is homozygous for a first allele or combination of alleles at said one or more genetic loci resulting in a first phenotype and said pollen parent is homozygous for a second allele or combination of alleles at said one or
• Hybrid seeds are generally produced by cross-pollinated plants.
• the term refers to the seed resulting from specific and controlled crossing of two inbred lines.
• the embryo of said hybrid seed comprises one haploid genome copy of each parent.
• inbred seed is produced when the egg inside the ovary of a plant is fertilized by pollen originating from anthers borne in the same flower (in cereals called spikelets) as the ovary, anthers from other flowers of the same plant or anthers from the flowers of another plant of the same parental origin.
• spikelets in cereals called spikelets
• the term "cereal” relates to members of the monocotyledonous family Poaceae which are cultivated for the edible components of their grain. These grains are composed of endosperm, germ and bran.
• Other members of the cereal family comprise rye, oats, barley, triticale, sorghum, wild rice, spelt, einkorn, emmer, durum wheat and kamut.
• a mixture of hybrid and inbred seed is obtained if the pollen parent is female fertile. Accordingly, a mixture of seeds comprising hybrid seeds can be obtained by interplanting a seed parent and a pollen parent. Possible scenarios of interplanting comprise strip or row planting where pollen and seed parent are planted in alternate rows.
• a more desirable type of interplanting comprises mixed interplanting, where a true mixture of pollen and seed parent is sown so that the pollen parent which is sown as a lower proportion than the seed parent is distributed evenly among the male sterile seed parent so that optimal cross-pollination can occur. This design is only feasible if the male sterile trait is inherent to the seed parent or can be induced due to a specific property of the seed parent.
• Male sterile in connection with the present invention refers to the failure of plants to produce functional anthers, pollen or male gametes. This can be due to natural or artificially introduced genetic predispositions or to human intervention on the plant in the field.
• Male fertile on the other hand relates to plants being capable of producing normal functional anthers, pollen and/or male gametes.
• a pollen parent is a parent plant that provides the male gametes (pollen) for fertilization, while a seed parent is the plant that provides the female gametes for fertilization, resulting in that plant bearing the seeds.
• Both seed and pollen parent should be planted so that they flower at the same time, i.e. pollen of the pollen parent and the ovules of the seed parent are ripe at the same time. If there are differences in flowering times, planting times are to be adapted accordingly.
• a seed parent can be male sterile and hence incapable of self-fertilization.
• a male-sterile seed parent can thus only produce seed when fertilized by pollen from a male fertile plant, i.e. the pollen parent.
• a pollen parent can be female sterile, thereby being incapable of self-fertilization.
• a female sterile pollen parent is thus only capable of fertilizing another female fertile plant, i.e. the seed parent.
• Reliably male sterile seed parents can be created in many crops. However, pollen parents in crops with "perfect" (bisexual) flowers, i.e. flowers comprising male and female organs are usually also female fertile and can therefore self-fertilize.
• the desired hybrid seed grows on the seed parent, whereas undesired contaminating inbred seed grows on the pollen parent.
• At least two types of seeds relates to different parental origin and hence genetic constitution (which may translate into differences in phenotype) of the seeds resulting from (cross-)fertilization of types of genetically equal or different parent plants.
• both parents are fertile, the following types of seeds can be produced; seeds resulting from self-fertilization of the first parent, seeds resulting from self- fertilization of the second parent and seeds resulting from cross -fertilization (i.e. hybrid seed) borne by either the first or the second parent.
• one parent being male sterile (i.e.
• Hybrid seed refers to seed resulting from a cross-fertilization of two parent plants that are not genetically identical, thus leading to seeds originating from the cross-fertilization having a different genetic constitution than either of the two parents.
• Different phenotypes allowing separation of hybrid seed with non-invasive methods based on the differences in said phenotypes relates to methods that do not require removing parts of the seed or damaging the seeds in any way to establish said phenotypic differences, thereby ensuring that the seeds, upon planting, germinate and develop normally.
• Such methods include separation based on relative size of the seeds by sieving, separation based on specific weight by gravity sorting on a table sorter, separation based on specific weight by wind sieving, separation based on differences in color using non-invasive imaging techniques.
• Such methods are for example described in Derne et al. (1997, Seeds handbook: biology, production, processing, and storage. Marcel Dekker, Inc., New York.), Harmond et al.
• the methods of the invention may comprise more than one separation step, for example two sieving steps with different cut-offs (see also Example 3).
• allele(s) means any of one or more alternative forms of a gene at a particular locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location or locus (plural: loci) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes or possibly on homeologous chromosomes.
• locus means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.
• homologous chromosomes means chromosomes that contain information for the same biological features and contain the same genes at the same loci but possibly different alleles of those genes.
• Homologous chromosomes are chromosomes that pair during meiosis.
• Non-homologous chromosomes representing all the biological features of an organism, form a set, and the number of sets in a cell is called ploidy. Diploid organisms contain two sets of non-homologous chromosomes, wherein each homologous chromosome is inherited from a different parent.
• tetraploid species two sets of diploid genomes exist, whereby the chromosomes of the two genomes are referred to as "homeologous chromosomes" (and similarly, the loci or genes of the two genomes are referred to as homeologous loci or genes).
• homeologous chromosomes the loci or genes of the two genomes are referred to as homeologous loci or genes.
• hexaploid species have three sets of diploid genomes, etc.
• a diploid, tetraploid or hexaploid plant species may comprise a large number of different alleles at a particular locus.
• heterozygous means a genetic condition existing when two different alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell.
• heterozygous means a genetic condition existing when two different alleles reside at a specific locus of the two members of a pair of homologous chromosomes in the cell.
• homozygous means a genetic condition existing when two identical alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell.
• homozygous means a genetic condition existing when two identical alleles reside at a specific locus on both members of a pair of homologous chromosomes in the cell.
• alternate alleles of one or more than one gene may determine the phenotype of the seed of inbred parent plants, whereby one allele or set of alleles determines one manifestation of the seed phenotype and another allele or set of alleles determines another manifestation of the seed phenotype and whereby one allele or set of alleles is dominant or partially dominant and the other allele or set of allelles is recessive or partially recessive.
• possible allele combinations specifying a particular seed phenotype include one dominant allele and one recessive allele as well as two dominant alleles, two recessive alleles, three dominant alleles, three recessive alleles, two dominant and one recessive allele, one dominant and two recessive alleles and all possible iterations for four, five, six or more genes involved in a specific phenotype according to the present invention. The same holds true for partially dominant and partially recessive alleles.
• the present invention relates to a method of producing hybrid seeds of a cereal comprising (a) interplanting a combination of plants comprising a male sterile or conditionally male sterile seed parent and a male fertile pollen parent, wherein said seed parent and pollen parent produce at least two types of seeds, at least one of which being said hybrid seeds, said at least two types of seed having different phenotypes allowing separation of said hybrid seed with non-invasive methods based on the differences in said phenotypes, said different phenotypes being determined by an allele or a combination of alleles at one or more genetic loci, wherein said seed parent is homozygous for a first allele or combination of alleles at said one or more genetic loci resulting in a first phenotype and said pollen parent is homozygous for a second allele or combination of alleles at said one or more genetic loci resulting in a second phenotype.
• said method of producing hybrid seeds of a cereal further comprises separating the hybrid seeds using a non-invasive separation method.
• said planting comprises row or strip planting or mixed interplanting.
• said planting comprises sowing the seed of or planting young plants of said seed parent and said pollen parent into the soil.
• said method of producing hybrid seeds of a cereal further comprises harvesting the seeds produced prior to separating.
• Harvesting may be effected, inter alia, by harvesting machines common in the art. In case of row or strip planting, harvesting machines are preferably designed so that the rows or strips of pollen plant can be harvested separately thus enabling for a pre-sorting of the resulting seeds.
• said seed parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles in said seed parent is dominant or partially dominant
• said pollen parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles in said pollen parent is recessive or partially recessive.
• partial dominance refers to the situation where the heterozygote displays a phenotype that is an intermediate between the phenotypes of both homozygote parent plants (one of which is homozygous (partially) dominant and the other is homozygous recessive). This intermediate phenotype is a demonstration of partial or incomplete dominance. When partial dominance occurs, a range of phenotypes is usually observed among the offspring. The same applies to partially recessive alleles. As used here, these terms imply that all alleles responsible for expression of hulled or free-threshing traits as explained further below, even if partially dominant or partially recessive, result in the expression of the desired phenotype, i.e.
• the above combination of the genetic constitutions of the parents results in two types of seed being generated, wherein said two types of seed can be separated based on phenotype, as described above.
• the seed parent is homozygous for one or more dominant alleles
• the resulting hybrid seed also carries the maternal (dominant) phenotype; either because the phenotype of the seed is directly determined by genotype of the plant bearing the seeds (in this case the seed parent), as will be explained further below, or because of the genotype of the seed itself determining the seed phenotype.
• the inbred seed resulting from self-fertilization of the homozygous recessive pollen parent carries the (recessive) paternal phenotype, either because the phenotype of the seed is directly determined by genotype of the plant bearing the seeds (in this case the pollen parent), or because of the genotype of the seed itself determining the seed phenotype.
• This difference in the genotype of the plant bearing the seeds determining the seed phenotype vs. the genotype of the seed itself determining the seed phenotype is referred to as maternal expression vs. endosperm/embryonic expression.
• said combination of plants is characterized in that said one allele or combination of alleles at one or more genetic loci in said seed parent manifests in seeds with a free-threshing phenotype and wherein said one allele or combination of alleles at one or more genetic loci in said pollen parent manifests in seeds with a hulled phenotype.
• Angiosperm seeds consist of three genetically distinct constituents: (1) the embryo formed from the zygote, (2) the endosperm, which is normally triploid, (3) the seed coat from tissue derived from the maternal tissue of the ovule.
• the seed coat is also referred to as husk or hull.
• the wheat ear breaks up into spikelets (also known as "speltoid phenotype").
• spikelets also known as "speltoid phenotype"
• further processing such as milling or pounding, is needed to remove the hulls or husks.
• Hulled wheat seeds are often stored as spikelets because the toughened glumes give good protection against pests of stored grain.
• free-threshing or naked
• the glumes are fragile and the rachis tough.
• the chaff breaks up, releasing the grains.
• the seed parent is homozygous for a dominant or partially dominant allele or a combination of such alleles conferring the free-threshing phenotype, while the pollen parent is homozygous for a recessive allele or a combination of such alleles conferring the hulled phenotype.
• the hybrid seed will also be free-threshing whereas the selfed (inbred) seed is non-free-threshing (hulled).
• the glume tenacity gene Tg along with the major domestication locus Q control threshability.
• the Q gene is a gene located on the long arm of wheat chromosome 5A (the Q locus) that is largely responsible for the domestication of wheat, as it confers the free threshing character by suppressing the speltoid phenotype.
• several positive pleiotropic effects are linked to Q, for example softening of the glumes, reduction of ear length, more spikelets per ear and toughness of the rachis, all of those features being favorable to harvesting (Salamini et al., 2002, Nature Reviews Genetics 3:429-441).
• the Q gene was cloned and molecularly characterized as an APETALA2 (AP2) like transcription factor.
• AP2 AP2
• the Q allele appeared to be more abundantly transcribed than q, and can be differentiated by an A/G SNP at position 2123 of the Q gene, leading to a single amino acid difference between the Q and q protein.
• the Q protein is characterized by an isoleucine at position 329, while q has a valine at this position, the latter amino acid appearing to limit homodimer formation.
• the effect of Q mainly depends on its dosage. Based on dosage experiments of Q and q alleles in various backgrounds it was established that q is hypomorphic to Q and 2.5 doses of q equaled 1 dose of Q.
• Tg tenacious glume
• Sog soft glume
• Sog is the main threshability gene in diploid wheat, the mutant allele (sog) being a recessive allele that confers a free threshing phenotype (Sood et al., 2009, Theor Appl Genet 199:341-351).
• the 2BS and 5AL QTL corresponded to the homoeologous genes Tg and Q of hexaploid wheats, respectively, where Tg2 the putative ortholog of Tg, was located on chromosome 2BS (hereinafter referred to as Tg 2B , Q 5A and Tg 2B ).
• Tg 2B chromosome 2BS
• Q 5A and Tg 2B chromosome 2BS
• free-threshing durum wheat thus can have the genotype tgtg 2B QQ 5A
• free-threshing bread wheat can have the genotype tgtg tgtg QQ ⁇
• hulled durum wheat can have the genoty e tgtg 2B qq 5A
• hulled bread wheat can have the genotype tgtg 2B tgt 2D qq 5A .
• kernel nakedness is a genetically complex trait; alleles at four different loci (designated as N-l , N-2, N-3 and N-4) are known to act epistatically to govern its expression.
• N-l is the major switch gene conditioning nakedness, and alleles of the other three loci interact with N-l and among each other to modify the degree to which nakedness is expressed.
• oat genotypes demonstrate different degrees of nakedness ranging from completely naked (or nearly naked) to partially naked, to partially covered, and all the way to completely covered phenotypes.
• a completely naked phenotype is expressed when dominant alleles are present at the N-l and N-2 loci, and either the dominant (N-3) or the recessive (n-3) allele is present at the third locus.
• Genotypes with homozygous recessive (n-l/n-1) alleles at the N-l locus will always have the covered phenotype regardless of the alleles present at the other loci.
• N-l/N-1 genotypes may have either naked or mosaic phenotypes depending on the alleles present at the other loci.
• the mosaic phenotype produces a mixture of naked and covered kernels, of various proportions, depending on the alleles present at the N-2 and N-3 loci.
• the N-4 locus reverses the effects of the other three loci. In a homozygous recessive (n-4/n-4) condition it is hypostatic to any dominant allele at the other three loci. When the N-4 locus is homozygous dominant (N-4/N-4) and the N-l locus is in a heterozygous condition, a covered phenotype is produced.
• An esterase isozyme marker was found to be tightly linked to one of the genes (presumably N-l) conditioning nakedness in oats (Kibite 2002, An Isozyme Marker linked to the N-l Gene Governing Nakedness in Oat, in: Oat newsletter Vol48).
• free-threshing oats thus can have the genotype N-l/N- 1 together with N-2/N-2 and N-3/N-3 or together with N-2/N-2 and N-3/n-3 and n-4/n-4, while hulled oats can have the genotype n-l/n-1 and N-4/N-4 or N-l/n-1 and N ⁇ l/N ⁇ l, where the status of N-2 or N-3 is irrelevant.
• the seed phenotype in terms of hulls is maternally determined as the seed coat is in fact maternal tissue. This means that the genotype of the parent plant bearing the seeds determines whether the seed borne by that plant (i.e. the Fl seed) is either hulled of free-threshing. Consequently, the genotype of the Fl seed parent determines the phenotype of the seed born by that plant, i.e. of the F2 seed, etc.
• the threshability phenotype of hybrid seed borne by the seed parent is determined by the genotype of the seed parent, while seed borne by the pollen parent as a result of self- fertilization of thus has the phenotype as determined by the genotype of the pollen parent.
• This principle of the genotype of plant bearing the seed directly determining the seed phenotype is referred to as maternal expression of the seed phenotype.
• said combination of plants is characterized in that said one allele or combination of alleles at one or more genetic loci in said seed parent manifests in seed with a hulled phenotype and wherein said one allele or combination of alleles at one or more genetic loci in said pollen parent manifests in seeds with a free-threshing phenotype.
• Barley is an exception to the above principle of the maternal expression of the seed threshability phenotype.
• barley In contrast to other cereals such as wheat and oats, barley typically has caryopses with adhering hulls at maturity, known as covered (hulled) barley.
• covered (hulled) barley A few barley cultivars however are a free-threshing, also called naked (hull- less) barley.
• covered barley a sticky adhesive substance appears 10 days after flowering on the caryopsis surface and that the substance is produced by the caryopsis (i.e.
• the covered/naked caryopsis in barley is controlled by a single locus (nud, for nudum) located on chromosome arm 7HL ;the covered caryopsis allele (Nud) being dominant over the naked one (nud).
• the Nud gene was identified as an ethylene response factor (ERF) family transcription factor gene.
• ERF ethylene response factor
• the naked/hulled phenotype of the seed is determined by the Nud genotype of the seed itself (i.e. embryo/endosperm determined) and not by the genotype of the seed parent (not maternally determined). This is referred to as endosperm/embryonic expression of the seed phenotype.
• hulled barley has a genotype of Nud/Nud or Nud/nud
• naked barley has a genotype of nud/nud.
• said seed parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles is recessive or partially recessive, and said pollen parent is homozygous for one or a combination of alleles at one or more genetic loci, wherein at least one of said alleles is dominant or partially dominant.
• the seed phenotype should be maternally expressed.
• said combination of plants is characterized in that said one allele or combination of alleles at one or more genetic loci in said seed parent manifests in seeds with a hulled phenotype and said one allele or combination of alleles at one or more genetic loci in said pollen parent manifests in seeds with a free-threshing phenotype.
• Said non-invasive separation method can be based on differences in size, surface condition or specific weight of said at least two types of seed as described above.
• separation can be effected based on different sizes of the grains with and without hulls. Whereas the grains themselves usually have the same or a comparable size, the presence of the hulls tightly attached to the grains of one phenotype makes the volume of this type of seed larger than that of the free-threshing grains alone.
• An alternative separation method may be based on specific weight of the seed. Hulled seed, due to its bigger volume which is partly filled only by air, has a lower specific weight than free-threshing seed.
• the present invention also relates to a method of separating hybrid seed from a mixture of hybrid seed and inbred seed comprising obtaining or providing said mixture, wherein embryos of said hybrid seeds have a genotype comprising one haploid genome copy of the seed parent and one haploid genome copy of the pollen parent, wherein embryos of said inbred seeds have a genotype comprising two haploid genome copies of the pollen parent, wherein said seed parent is male sterile or conditionally male sterile and wherein said pollen parent is male fertile, characterized in that (a) said seed parent is homozygous for seed with a free-threshing phenotype and said pollen parent is homozygous for seed with a hulled phenotype, or (b) said seed parent is homozygous for seed with a hulled phenotype and said pollen parent is homozygous for seed with a free-threshing phenotype; comprising applying said mixture of seeds to a means for separation according to size, surface condition
• ratios of pollen parent to seed parent may be necessary.
• the ratio of pollen parent to seed parent may be ranging from 10:100 to 25:100, such as 15:100 or 20:100.
• less pollen parent is needed to ensure fertilization of the seed parent.
• ratios range between 1 :100 and 15:100, such as 2:100, 3:100, 5:100, 8:100, 10:100 or 12:100.
• Said cereal may be selected from wheat, durum, oat, barley and triticale.
• said cereal is wheat and said seed parent is selected from Triticum aestivum L. ssp. Vulgare Host, Triticum aestivum L. ssp. Compactum, Triticum aestivum L. ssp. Sphaerococcum, Triticum aestivum L. ssp. Petropavlovskyi and Triticum aestivum L. ssp. (2015)ianum.
• said cereal is wheat and said pollen parent is selected from Triticum aestivum L. ssp. Spelta, Triticumaestivum L. ssp. Macha,Triticum aestivum L. ssp. Vavilovii, Triticum aestivum L. ssp. Yunnanense and a semi-wild form of Triticum aestivum L. ssp. (2015)ianum.
• the cereal is wheat and seed parent and pollen parent are combined from the above listed cultivars.
• said cereal is durum wheat and said seed parent is selected from T. turgidum L. ssp. Durum Desf. And T. turgidum L. ssp. Parvicoccum.
• said cereal is durum wheat and said pollen parent is selected from T. turgidum L. ssp. Dicoccum (emmer) and T. turgidum L. ssp. Dicoccoides (wild emmer).
• the cereal is durum wheat and seed parent and pollen parent are combined from the above listed cultivars.
• the genes determining said hulled phenotype are recessively inherited.
• said male sterility is based on cytoplasmic male sterility, nuclear male sterility or a trait introduced into said plant by genetic engineering or mutation, or induced using a gametocide.
• CMS Cytoplasmic male-sterile
• the seed parent For cytoplasmic male sterility to be used in hybrid seed production, the seed parent must contain a sterile cytoplasm and the pollen parent must contain (nuclear) restorer genes to restore the fertility of the hybrid plants grown from the hybrid seed harvested from the seed parent; and the CMS mutation should not be associated with a yield penalty.
• Biotechnology has added new possibilities to obtain male-sterile plants, i.e. by introducing genes resulting in traits that confer (conditional) male sterility.
• a gene, or set of genes may be introduced via genetic engineering, said genes being able to alter the levels of metabolites needed for the production of viable pollen, such as amino acids (W094/29465), sugars (Goetz et al., 2001, PNAS 98, 6522-6527), flavonols (Derksen et al., 1999, Planta 207, 575-581), jasmonic acid (WO97/10703, McConn and Browse, Plant Cell 8, 403416; Sanders et al., Plant Cell 12, 1041-1061), biotin (WO99/04023) or auxins (Spena et al., 1992, Theor.
• Another approach to general male sterility is effected by the combined action of two (trans)genes brought together into the same plant by crossing two different parental lines each expressing one of the genes (see. E. g. US 5,426,041 or W098/32325).
• An example of this approach is the reconstitution of an active toxin from two inactive toxin fragments each encoded by a separate transgene.
• the combined expression of both transgenes in the male reproductive tissues can result in male sterility.
• This strategy requires a pollination control system to cross the two fertile grandparent lines to produce the male-sterile female parent line for hybrid seed production.
• Systems that rely on the selective removal of tissues needed for the production of functional pollen by the tissue- specific expression of a gene encoding a protein able to disrupt cell function can be based on both natural traits and genetically engineered traits (see. e. g. Mariani et al., 1990, Nature 347, 737-741; Koltunow et al, 1990, Plant Cell 2, 1201 -1224; US 6,072,102 and US5,880,333). Some of these offer the possibility for chemical control of sterility or of fertility. For example, inducible sterility can be obtained by expressing a gene encoding a protein that catalyzes the conversion of a pro-herbicide into a herbicide only in male reproductive tissues (see e. g.
• the present invention also relates to hybrid cereal seed produced by the methods of the invention as described above.
• the present invention relates to hybrid cereal seed, wherein embryos of said hybrid seeds have a genotype comprising one haploid genome copy of the seed parent and one haploid genome copy of the pollen parent, optionally wherein said seed parent is male sterile or conditionally male sterile, wherein said pollen parent is male fertile, characterized in that (a) said seed parent is homozygous for seed with a free-threshing phenotype and said pollen parent is homozygous for seed with a hulled phenotype; or b) said seed parent is homozygous for seed with a hulled phenotype and said pollen parent is homozygous for seeds with a free-threshing phenotype.
• the present invention also relates to the use of (a) a male sterile or conditionally male sterile seed parent producing seeds with a free-threshing phenotype of a cereal and a pollen parent homozygous for seed with a hulled phenotype of said same cereal, or (b) a male sterile or conditionally male sterile seed parent of a cereal producing seed with a hulled phenotype and a pollen parent homozygous for seed with a free-threshing phenotype for producing hybrid seed of said cereal.
• the present invention also relates to hybrid seed obtained by any of the methods described herein.
• Example 1 Separation of artificial seed mixtures
• Table 4 shows the target fractions after sieving a sample of 100 g.
• the number of hulled and free threshing seeds was counted in the target fraction. It was possible to enrich the target fraction in all species and environments higher than the required threshold of 90%.
• the purity of the target fraction varied from 90% to 95%.
• the variation of the purity in oats varied between 94% and 96%.
• the best results for separating hulled and free threshing seeds could be found for wheat.
• the purity of the target fraction in wheat was in both entries Badenstern-Tabasco and Badenkrone-Genius more than 99%.
• Table 5 shows the purity of the target fraction before and after sieving the mixed samples.
• Table 5 Efficiency of sieving a mixture of hulled and free threshing seed sample in different species
• the hulled and free threshing seed show different colors.
• the difference in seed color would allow the seed processor to remove further pollen parent seeds by color sorting machines.
• By combining simple sieving techniques with standard color sorting techniques it is possible to enrich the fraction of Fl seeds harvested from a mixed planting to more than 99%.
• Hulled and free threshing commercial wheat varieties (Table 6) were grown at the location D-78224 Bohlingen. The field design is shown in table 7.
• row planting is necessary. The seed parent row was flanked by rows of the pollen parent. As this was a small scale demonstration, rows of 1 ,0 m length were planted with a plant breeder's sowing machine. One plot therefore consisted of three rows.
• male sterility of the seed parent was introduced via treatment with a gametocide (e.g. Genesis) according to the gametocide's instruction manual.
• Successful introduction of male sterility of the seed parent was checked by bagging some single ears within the seed parent row. Bagged single ears showed no seed set. All three rows were harvested together. Harvested seed was cleaned from dirt and other unwanted material by wind sieving.
• Example 3 Production of wheat hybrid seed by using hulled pollen parents, where the seed parent exhibits cytoplasmatic male sterility (CMS) and is a free threshing hexaploid winter wheat.
• CMS cytoplasmatic male sterility
• CMS-WW005 Triticum aestivum
• This line exhibits cytoplasmatic male sterility (CMS).
• CMS cytoplasmatic male sterility
• Hybrid production was performed in mixed planting in the greenhouse at Gatersleben. Seed parent was pollinated by open pollination with the pollen parents of the variety Badenstern or Badenkrone (Triticum spelta). Badenstern or Badenkrone are fully fertile pollen parents with non-free threshing (hulled) grain set. Another hybrid seed production was performed in mixed planting in the counter season nursery in Pochefstroom /Rep. South Africa. Mixing rate was 50% seed parent and 50% pollen parent in order to have enough pollen. Plot size at all locations was 1 m * 1.2 m, with four rows in Gatersleben and three rows in Pochefstroom. Table 1 : parents
• Sample 4 i.e. the harvest from the production of CMS-WW005 * Badenkrone at Pochefstroom, did not contain Fl seeds but only inbred seeds from the pollen parent, as the nicking was insufficient (flowering time of seed and pollen parent differed too much) and seed parent plants were not pollinated. Sample 3 only contained inbred seed from the pollen parent Badenstern and was harvested in Gatersleben for comparison purposes.
• OECD SEED SCHEMES 2014 OECD Schemes for the Varietal Certification or the Control of Seed Moving in International Trade, ANNEX VIII, pages 99-101, OECD 2014, http:// ⁇ vww.oecd.org/tad/code/l_complete%i20document.pdf).
• Table 2 Sorting of harvested Fl seed mixtures.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Environmental Sciences (AREA)
• Developmental Biology & Embryology (AREA)
• Botany (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Physiology (AREA)
• General Health & Medical Sciences (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Cell Biology (AREA)
• Plant Pathology (AREA)
• Natural Medicines & Medicinal Plants (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Pretreatment Of Seeds And Plants (AREA)

Priority Applications (8)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (1)

Family

ID=47901820

Family Applications (1)

Country Status (9)

Cited By (3)

Families Citing this family (2)

Citations (7)

Family Cites Families (14)

• 2013
  • 2013-03-18 EP EP13159765.0A patent/EP2781151A1/en not_active Withdrawn
• 2014
  • 2014-03-14 AR ARP140101110A patent/AR095505A1/es unknown
  • 2014-03-17 EP EP14710304.8A patent/EP2975927B1/en active Active
  • 2014-03-17 PL PL14710304T patent/PL2975927T3/pl unknown
  • 2014-03-17 AU AU2014234456A patent/AU2014234456B2/en active Active
  • 2014-03-17 BR BR112015023991A patent/BR112015023991A2/pt not_active Application Discontinuation
  • 2014-03-17 CA CA2906998A patent/CA2906998C/en active Active
  • 2014-03-17 WO PCT/EP2014/055249 patent/WO2014147005A1/en active Application Filing
  • 2014-03-17 UA UAA201510077A patent/UA120163C2/uk unknown
  • 2014-03-17 US US14/772,518 patent/US10028460B2/en active Active
• 2018
  • 2018-06-26 US US16/018,103 patent/US10743486B2/en active Active

Patent Citations (7)

Also Published As

Similar Documents

Legal Events